A retrovirus designated human immunodeficiency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the extensive post-translational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Inhibition of this processing prevents the production of normally infectious virus. For example, Kohl, N. E., et. al., Proc. Natl. Acad. Sci. USA, 85, 4686 (1988), demonstrated that genetic inactivation of the HIV encoded protease resulted in the production of immature, non-infectious virus particles. These results suggest that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame [Ratner, L. et al., Nature, 313, 277 (1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an endonuclease and an HIV protease [Toh, H. et al., EMBO J. 4, 1267 (1985); Power, M. D. et al., Science, 231, 1567 (1986); Pearl, L. H. et al., Nature 329, 351 (1987)]. Applicants construct a vector and expression system for HIV protease. Related an includes Baum, E. Z. et al., Proc. Natl. Acad. Sci. 87, 10023 (1990).
The particular advantages of the present invention include the coordinate expression of functional HIV protease and a reporter beta-galactosidase having an insert cleavable by the HIV protease. The coordinate expression results from the transcription of a single dicistronic mRNA. Control over the expression of enzyme (HIV protease) and substrate (the cleavable insert) is readily achieved with this type of recombinant construction.
Further, the present invention is directed to a rapid method of identifying drug-resistant HIV protease mutants. Because only the HindIII site on the 5' side of the .beta.-galactosidase gene is reconstructed in the cloning, the HIV protease gene is flanked by unique NdeI and HindIII sites, enabling easy removal and insertion of alternate protease genes. Thus, libraries of mutagenized protease genes for screening are constructed and inserted into this vector as NdeI-HindIII fragments.
Finally, because the promoter controlling the coordinate expression is itself regulatable, manipulation of the internal concentration of HIV protease is achieved. This arrangement avoids the toxic effects of intracellular HIV protease. Applicants induce the regulatable promoter, here the tryptophan promoter, only when further growth of the host cell E.coli is no longer needed.